The interactions of leptin with the blood-brain barrier (BBB) play an important role in some forms of obesity. In this proposal, we will elucidate the novel aspects of how leptin regulates the permeation of some feeding-related peptides and cytokines across the BBB. (a) to test the hypothesis that leptin recruits receptors of selective satiety peptides and activates transport, we will examine how leptin increases the blood-to-brain permeation of urocortin, another potent satiety peptide. We will first determine whether receptors for both leptin and urocortin are involved in this process. We will then determine whether there is heterodimerization of these receptors by use of co-immunoprecipitation and ligand binding assays, after the receptors are expressed by co-transfection on mouse TM-BBB4 brain endothelial cells. Further, we will determine by immunofluorescent microscopy whether there is co-localization of these receptors on endocytotic vesicles after binding to leptin. (b) To test the hypothesis that leptin can enhance an existing transport system for a cytokine that also reduces feeding, we will examine how leptin upregulates the transport of tumor necrosis factor alpha (TNFalpha). We predict that leptin will crease TNFalpha influx by modulating phosphorylation of the p55-receptors, p75-receptors, and proteins related to TNFalpha transport, as shown by transport assays, immunoprecipitation, and Western blot. (c) To test the physiological relevance of leptin-mediated regulation of TNFalpha and urocortin transport, we will measure the transport of TNFalpha, urocortin, as well as leptin itself in mice that are fed normally and in mice that are food-deprived. We predict that leptin will increase urocortin and TNFalpha transport only in mice with free access to food, thereby providing additional satiety signals to the brain when leptin is already relatively high in the circulation. In contrast, we predict that in food-deprived mice, the transport systems for both leptin and TNFalpha will be down-regulated and that for urocortin will not be activated, as expected for anorectic agents under the severe condition in which no food is available. By completing the proposed studies, we will demonstrate the presence of novel protein-protein interactions at the BBB, the mechanisms involved in these interactions, and the additional functional role of the BBB in regulating feeding. This information will not only add to a better understanding of the mechanisms of BBB transport in general but also to the potential therapeutic use of ingestive peptides.